Specific binding assay conjugate with spaced-apart receptors

ABSTRACT

In the immunoassay of antigens in liquids, a reaction mixture is formed containing the liquid under assay, labelled antigen, and a mixed binding reagent which contains an antigen-binding site and a label-binding site, the two sites being spaced apart in the reagent so that a single molecule of labelled antigen cannot bind to both sites. The label is one whose activity is changed upon binding to a label-binding site, and the amount of antigen in the original liquid sample is determined by measuring the activity of the label in the reaction mixture. A preferred label is a fluorophore. The mixed binding reagent preferably consists of two antibodies linked together.

This application is a division of U.S. Pat. No. 4,401,764 issued Aug.30, 1983.

This invention relates to the immunoassay of antigens (which termincludes haptens), using a labelled reagent.

Immunoassays which utilise the immunospecific reaction between anantibody and an antigen are well known. It is also known to use in suchassays a labelled reagent, part of which becomes bound and part remainsfree in the reaction mixture, and to determine quantitatively theantibody or antigen under assay by measuring the amount of free or boundlabelled reagent. In certain assays of this type, such asradioimmunoassays, it is necessary to separate the bound labelledreagent from the free labelled reagent before measuring the amount oflabel, and this separation step is not only time-consuming but can be asource of error. Attention has therefore been focussed in recent yearson providing an immunoassay in which a separation step is unnecessary,and several workable proposals have been made, including some utilisinga fluorophore label.

In fluoroimmunoassays, the amount of bound or free fluorophore-labelledreagent is measured by fluorescence. In the fluoroimmunoassays ofcertain antigens, the fluorescence of the labelled reagent changes whenit becomes bound, so that a separation step is then unnecessary. Such anassay is described in our U.S. Pat. No. 4,150,949 and may be used toassay haptens such as gentamicin. This change in fluorescence does notoccur with all antigens, however, and in an alternative approach, thefluorescence of the free fluorophore-labelled reagent in the assaymixture is quenched by introducing an antibody to the fluorophore-label.Thus the antigen to be assayed is mixed with fluorophore-labelledreagent and with antibody to the antigen, and part of the reagentbecomes bound and part remains free in the mixture. Antibody to thefluorophore is then added, which binds to the free reagent and changes(usually quenches) the fluorescence thereof. The net resultingfluorescence of the mixture gives a measure of the amount of labelledreagent originally bound to the antibody against the antigen.

Whilst this procedure is satisfactory with certain antigens, the problemarises that in some cases, the antibody to the label will bind not onlywith the free labelled reagent but also with the bound labelled reagent.We have now devised a way of overcoming this problem.

In one aspect, the present invention provides a method of immunoassay ofan antigen (Ag) in a liquid sample, which comprises forming a mixtureof:

(a) sample;

(b) a substance bearing a non-radiosotopic label, which substance is thesame as the Ag under assay or is so closely similar as to be bindable byan antibody against the Ag;

(c) an antibody against the Ag; and detecting the amount of labelactivity in the mixture and therefrom determining the amount of Ag;characterised in that:

component (c) is a mixed binding reagent which comprises at least onefirst site selectively bindable to the Ag, and at least one second siteselectively bindable to the label, the first and second sites beingspaced apart so that a single molecule of labelled substance cannotsimultaneously become bound to both a first and a second site, andwherein the activity of the label is changed upon binding to the secondsites.

The method of the invention is applicable to assays utilising labelssuch as fluorophores (or potential fluorophores), spin labels,chemiluminescent labels and, in certain circumstances, enzyme orcoenzyme labels. Preferably, however, a fluorophore (or potentialfluorophore) label is used and the invention will hereafter be describedprincipally with reference to fluoroimmunoassays, it being understoodthat in principle and mutatis mutandis other labels such as describedmay be used. By "potential fluorophore label", we mean a label whichdoes not normally fluoresce but which can be made to fluoresce.

An important feature of the invention is the mixed binding reagent(hereinafter MBR) utilised in step (c). This reagent will normallycomprise two antibodies (one against the antigen under assay and theother against the label), the two different antibodies being linkedtogether to form a unitary reagent complex in which the first and secondbinding sites are spaced apart so that each molecule of the labelledsubstance can only bind to one site and not simultaneously to both.Thus, in the assay of the invention, the problem encountered in theprior art of a molecule of the labelled substance becoming bound to bothtypes of antibody, is overcome.

The assay of the invention proceeds generally as follows. The samplecontaining the antigen under assay is mixed with the labelled substanceand the MBR. The antigen and the labelled substance compete for thefirst (i.e. antigen-binding) sites in the MBR. The fluorescence of thelabelled reagent is preferably such that it does not changesignificantly when the substance binds to a first site in the MBR.

The portion of labelled reagent which does not bind to a first site inthe MBR is available to bind, via its label, to a second (i.e.label-binding) site in the MBR whereupon its fluorescence will changemarkedly. Overall, the net resulting fluorescence of the mixture willvary with the amount of Ag present in the original sample, since thisaffects the amount of labelled reagent both remaining free and becomingbound to second sites in the MBR. By first preparing standard resultsrelating fluorescence to antigen concentration, the amount of antigen inany sample can be determined.

The MBR is an important feature of the present invention. It comprisestwo different types of binding sites which are spaced apart so as toprevent a molecule of the bifunctional labelled substance becoming boundto both types of sites. The binding sites themselves are most preferablyprovided by antibodies or by fragments of antibodies containing theirbinding sites, although one or both sites may be provided in the form ofactive chemical groups other than an antibody. A convenient andpreferred MBR comprises an antibody to the antigen (to provide firstsites), and an antibody to the label (to provide second sites), the twoantibodies being linked together in a manner such as to achieve thedesired spacing. In one convenient type of MBR, the two antibodiesproviding the binding sites are linked by a "second" antibody. Thus, thetwo binding site-antibodies are raised in the same animal species (e.g.rabbit) and are then linked by an antibody (the "second" antibody) tothe immunoglobulin of that species (e.g. by sheep (anti-rabbit Ig)antibodies). In this way, the two rabbit-generated antibodies are solocated that their respective binding sites for the antigen and labelare spaced so that a molecule of labelled substance cannotsimultaneously bind to both sites.

The second antibody is only one of the various ways in which theantigen-binding and label-binding antibodies (or other bindingsubstances) can be linked to form an MBR Another possibility is to useprotein A in place of the second antibody. Alternatively, both theantigen-binding and the label-binding antibodies can be conjugated witha hapten (or, more generally, an antigen), and then bound by theaddition of an antibody to the hapten. Using this technique, it is notnecessary for the antigen-binding and label-binding antibodies to havebeen raised in the same animal species.

Another possibility is for the two antibodies to be linked to a solidsubstrate. A particularly preferred such substrate is polyacrylamidebeads which are known for use in fluoroimmunoassays, and which have aspecific gravity close to 1 and also a refractive index close to that ofwater so that they do not seriously interfere with the fluorescencemeasurement by light scattering. Such beads are commercially available.By activating the surfaces of the beads, the two antibodies can becovalently or otherwise bound thereto to provide an MBR for use in thepresent invention.

Another way of preparing an MBR is to link the two antibodies togetherusing a bifunctional chemical bridging group of an appropriate kind,many of which are well known in protein chemistry.

The preferred fluorophore labels are fluorescein, dansyl, rhodamine,fluorescamine, pyrene, 2-methoxy-2,4-diphenyl-3(2H)-furanone, andumbelliferone and derivatives thereof. Of these, fluorescein is the mostpreferred. The label must be one whose activity (e.g. fluorescence) ischanged upon binding to a second site.

The method of the present invention is widely applicable to the assay ofantigens, but it is particularly of interest in the assay of haptens,such as gentamicin and similar aminoglycoside antibiotics, and phenytoinand nortriptyline and similar drugs. It can also be used for the assayof thyroid hormones such as thyroxine (T₄), although normally in theassay of human sera for T₄ or nortriptyline, there will be significantfluorescence interference unless the serum is first treated to removethe interferants.

The nature of the labelled substance is not critical except that it mustbe bindable by the antibody against the antigen under assay, and alsothe fluorescence (or other activity) of the label must change when thelabel binds to the anti-label binding sites in, for example, anantibody. Generally, the substance will be the same as the antigen underassay, but where it is difficult to bind a label to the antigen, thenanother substance is used which is sufficiently similar to the antigento bind to the antigen binding sites in the MBR.

The method of the invention may be carried out on a discrete manualbasis, or in an automated fashion on a plurality of samples using, forexample, the well known continuous flow techniques. In continuous flowanalyses according to the present invention, the mixture of sample,fluorescent labelled substance and MBR, is passed along a conduit andthe fluorescence is then measured. In a preferred procedure, which isdescribed in U.S. Pat. No. 2,797,149, individual segments of mixture arepassed sequentially along the conduit, separated by an inert fluidsegment (e.g. air) and, if desired, a wash liquid segment. The mixturecan be formed in the conduit itself, by supplying to the conduit, inphase with segments of components of the mixture already presenttherein, the one or more further components, mixing of the componentsoccurring in the conduit as the mixture flows therethrough.

The following experimental results illustrate the method of theinvention.

1. Assay of thyroxine (T₄)

MATERIALS

L-T₄, as the free acid, was obtained from Sigma and labelled withfluorescein isothiocyanate as described in our copending U.K.application No. 38710/76 Rabbit anti-fluorescein serum and sheepanti-rabbit immunoglobulin G serum were prepared by standard methods.Rabbit and sheep anti-T₄ sera were obtained from Dr. G. Zborowski(Technicon Instruments Corp., Tarrytown, N.Y., U.S.A.) and Dr. T. G.Merrett (Benenden Chest Hospital, Cranbrook, Kent, U.K.), respectively.

All experiments were performed using 75 mmol/l barbital buffer, pH 8.6at ambient temperature.

METHODS Fluorimetry

Fluorescence was measured using a Perkin-Elmer Model 1000 fluorimeter,fitted with appropriate filters. In the experiments a correction wasmade for the background signal contributed by reactants other thanfluorescein-labelled T₄. This was determined by fluorimetry ofincubation mixtures containing no labelled hormone. Results wereexpressed relative to an arbitrary scale of fluorescence intensity.

Fluorescence of labelled T₄ in presence of antibody excess

To 500 μl of fluorescein-labelled T₄ (30 μg/l) was added 500 μl ofantiserum, control serum or buffer, followed after at least 5 min by theaddition of 500 μl of a different antiserum, control serum or buffer.Fluorescence was determined as above. Rabbit anti-fluorescein and rabbitcontrol serum were present at a final dilution of 1:6400 and sheepanti-T₄ serum and sheep control serum at a final dilution of 1:400.

Fluorescence of labelled T₄ in the presence of doubling antiserumdilutions

To 500 μl aliquots of doubling dilutions of rabbit anti-T₄, sheepanti-T₄, rabbit anti-fluorescein and control sera from sheep and rabbitswas added 1 ml of fluorescein-labelled T₄ (15 μg/l). Fluorescence wasdetermined after an incubation period of at least 5 min.

Formation of mixed binding reagent

To 500 μl aliquots of doubling dilutions of sheep anti-rabbitimmunoglobulin G serum was added 500 μl of a mixture of rabbit anti-T₄and rabbit anti-fluorescein serum. After 60 min, 500 μl offluorescein-labelled T₄ (30 μg/l) was added and the fluorescein measuredas above.

Fluoroimmunoassay of the invention using a mixed binding reagent

The mixed binding reagent was first prepared as above. To 100 μl ofaliquots of standard solutions of T₄ in buffer was added 650 μl offluorescein-labelled T₄ (23 μg/l) followed by 750 μl of the mixedantibody complex and the fluorescence was then determined as above. Acontrol experiment was performed in an identical manner except that asheep control serum was used in place of the sheep anti-rabbitimmunglobulin G serum.

RESULTS Fluorescence of labelled T₄ in the presence of antibody excess

The results are summarised in Table 1. Addition of sheep anti-T₄ serumin place of buffer resulted in the expected enhancement of fluorescence(from 16 to 42 fluorescence units) which was not affected significantlyby the presence of rabbit control serum. Conversely, addition of rabbitanti-fluorescein serum in place of buffer caused a marked decrease influorescence (from 16 to 2 fluorescence units) unaffected by sheepcontrol serum. Irrespective of the order of addition, the fluoresence offluorescein-labelled T₄ was largely quenched in the presence of both theanti-T₄ and anti-fluorescein sera, relative to the signal when bound byantibodies to T₄ alone. In all cases the reaction was complete within 1min as judged by attainment of stable fluorescent read-out.

Fluorescence of labelled T₄ in the presence of doubling antiserumdilutions

Rabbit anti-T₄ and rabbit anti-fluorescein dilution curves were obtainedin order to choose appropriate dilutions of these antisera for use informing the mixed binding reagent. A sheep anti-T₄ dilution curve wasalso obtained and the results are shown in FIG. 2, together with theresults using control rabbit and sheep sera. Binding offluorescein-labelled T₄ by antibodies to T₄ and to fluorescein resultedin the expected enhancement and quenching of fluorescence, respectively.Non-specific effects were negligible. On the basis of these studies afinal dilution of rabbit anti-T₄ of 1:1600 and of rabbitanti-fluorescein of 1:6400 was chosen to form the mixed antibodyreagent.

Formation of mixed binding reagent

The fluorescence of fluorescein-labelled T₄ added to a mixture of rabbitanti-T₄ and anti-fluorescein sera was increased some two-fold by priorcomplexation of the antibodies with sufficient amounts of sheepanti-rabbit immunoglobulin G serum (FIG. 3). On the basis of thisexperiment a final dilution of sheep antiserum of 1:160 was chosen toform the mixed binding reagent. Prior incubation of the two rabbitantisera with serum from a control sheep had no significant effect.

Fluoroimmunoassay of the invention using mixed binding reagent

Using the mixed binding reagent, a standard curve for T₄ was obtained(FIG. 4) with the addition of increasing amounts of unlabelled T₄causing a progressive decrease in the final fluorescence reading Thecontrol experiment confirmed the dependence of the observed effects onthe presence of specific sheep anti-rabbit immunoglobulin G serum.

The background signal from reagents other than the labelled T₄ was 10units and was largely contributed by the intrinsic fluorescence of therelatively high concentration of sheep antiserum present. The standardcurve obtained employing a mixed binding reagent 24 h after preparationwas the same as that obtained after 60 min although the mixed bindingreagent had developed visible turbidity and the background signal hadincreased to 12 fluorescence units.

                  TABLE 1                                                         ______________________________________                                        Fluorescence of Fluorescein-labelled                                          T.sub.4 in the Presence of Antibody Excess                                    Order of Reagent Addition to Labelled T.sub.4                                                          Fluorescence                                         First Reagent Second Reagent Intensity                                        ______________________________________                                        Buffer        Buffer         16                                               Anti-T.sub.4 serum*                                                                         Buffer         42                                               Anti-T.sub.4 serum                                                                          Rabbit control serum                                                                         41                                               Anti-fluorescein serum                                                                      Buffer          2                                               Anti-fluorescein serum                                                                      Sheep control serum                                                                           2                                               Anti-fluorescein serum                                                                      Anti-T.sub.4 serum                                                                            6                                               Anti-T.sub.4 serum                                                                          Anti-fluorescein serum                                                                       10                                               ______________________________________                                         *The antiserum dilutions employed are given in the methods section       

2. Assay of Amikacin

MATERIALS

The following reagents were used: rabbit anti-fluorescein serum; rabbitanti-amikacin serum; sheep anti-rabbit immunoglobulin G serum; andfluorescein-labelled amikacin prepared by the reaction of amikacin withfluorescein isothiocyanate. The buffer used was 100 mmol/l sodiumphosphate, pH 7.5, containing 1 ml/l Triton X-100 detergent and 1 g/lsodium azide.

Formation of mixed binding reagent

To a mixture of anti-fluorescein serum and anti-amikacin serum was addedanti-rabbit immunoglobulin G serum, so as to give final dilutions of theantisera as follows: anti-amikacin 1:320; anti-fluorescein 1:2,000; andanti-rabbit immunoglobulin G 1:40. The mixture was left at roomtemperature for 1 hour before use.

Assay procedure

To 50 μl aliquots of standard solutions of amikacin in buffer was added750 μl of fluorescein-labelled amikacin (20 nmol/l concentrationestimated spectrophotometrically), followed by 750 μl of mixed bindingreagent. After incubation for 30 min at room temperature, thefluorescence of the mixtures was determined. Correction was made for thebackground signal contributed by reactants other thanfluorescein-labelled amikacin; this was determined by measurement of thefluorescence of mixtures containing no labelled antibiotic. Results wereexpressed relative to an arbitrary scale of fluorescence intensity. Thestandard curve obtained is shown in FIG. 5.

IN THE ACCOMPANYING DRAWINGS

FIGS. 1A and 1B are a diagrammatic representation of one embodiment ofthe method of the invention as applied to fluoroimmunoassays. In FIGS.1A and 1B, the mixed binding reagent 1 has anti-hapten binding sites 2and anti-label binding sites 3. In FIG. 1A, the hapten 5 carries afluorescein label 6. In FIG. 1B, some of the hapten 5 is labelled andsome is not. In FIG. 1A, when the labelled hapten is incubated with theMBR 1, some of the labelled hapten will be bound to the anti-haptensites 2 and continue to fluoresce, while some will be bound by theanti-fluorescein sites 3 with a resultant decrease in fluorescence. InFIG. 1B unlabelled hapten is also present in the reaction mixture. Thiscompetes with labelled hapten for anti-hapten binding sites 2, and asmore labelled hapten becomes bound to anti-fluorescein sites 3, therewill be a further decrease in fluorescence. Thus the fluorescence of theincubation mixtures at equilibrium will be inversely related to theinitial amount of unlabelled hapten present.

FIG. 2 shows anti-T₄ and anti-fluorescein dilution curves. Closedcircles, sheep anti-T₄ serum; open circles, rabbit anti-T₄ serum; closedtriangles, control sheep serum; open triangles, control rabbit serum;closed squares, anti-fluorescein serum.

FIG. 3 is an anti-immunoglobulin G dilution curve in formation of mixedantibody reagent. Final dilutions: anti-T₄ serum 1:1600;anti-fluorescein serum 1:6400. Open circle shows fluorescence in absenceof added anti-immunoglobulin G serum.

FIG. 4 is the fluoroimmunoassay standard curve. Closed circles, usingmixed binding reagent; open circles, anti-immunoglobulin G serumreplaced by control sheep serum. Final dilutions: anti-T₄ serum 1:1600;anti-fluorescein serum 1:6400; anti-immunoglobulin G serum or controlsheep serum 1:160.

FIG. 5 is the fluoroimmunoassay standard curve for the assay ofamikacin.

I claim:
 1. A specific binding assay conjugate which comprises at leastone analyte-specific receptor linked with a label-specific receptor inspaced relationship through at least one linking group such that alabeled analyte or analyte analog cannot simultaneously bind with theanalyte-specific receptor and the label-specific receptor.
 2. Thespecific binding assay conjugate of claim 1 wherein the analyte-specificreceptor is linked with the label-specific receptor through at least oneprotein A molecule.
 3. The specific binding assay conjugate of claim 1wherein the analyte-specific receptor and the label-specific receptorare linked to a solid substrate.
 4. The specific binding assay conjugateof claim 1 wherein said label is detectably modifiable by binding withthe receptor therefor.
 5. The specific binding assay conjugate of claim1 wherein the analyte-specific receptor and the label-specific receptorare antibodies and are linked by an antibody.
 6. The specific bindingassay conjugate of claim 5 wherein said analyte specific antibody andlabel-specific antibody are from a first animal species and said linkingantibody is from a second animal species and is specific to bind withantibodies of said first animal species.
 7. The specific binding assayconjugate of claim 1 wherein the analyte-specific receptor and thelabel-specific receptor are each conjugated with an antigen and theantigen of each of said conjugates is bound to a common antibodytherefor.
 8. The specific binding assay conjugate of claim 7 wherein theanalyte specific receptor and the label-specific receptor are eachantibodies.
 9. The specific binding assay conjugate of claim 8 whereinsaid analyte specific antibody and said label-specific antibody are eachfrom the same animal species.
 10. The specific binding assay conjugateof claim 8 wherein the analyte specific antibody and the label-specificantibody are each from an animal species different from the other.